Cardiovascular disease accounts for an overwhelming proportion of the morbidity and mortality suffered by patients with all forms of diabetes. Hyperglycemia is considered an important etiologic factor that serves as the initial trigger for diabetic vascular complications. Clinical evidence demonstrates that cardiovascular disease in diabetic patients increases as a function of the duration of hyperglycemia and that exposure of the vasculature to elevated ambient glucose causes generalized endothelial dysfunction, accelerates the atherosclerotic process, and impairs important functions of the microcirculation. As a consequence of hyperglycemia, the diabetic vasculature experiences abnormal inflammatory processes characterized by impaired release of nitric oxide (NO) and increased endothelial adhesiveness. Calpains are a family of calcium-dependent proteases, which have been recently implicated in acute inflammatory disorders of the cardiovascular system. In preliminary studies, we have made the novel observation that inhibition of calpain activity preserves release of endothelial NO and attenuates inflammatory leukocyte-endothelium interactions in the microcirculation during hyperglycemia. Our data strongly support a role for calpains in the pathophysiology of diabetic vascular disease, suggesting a potentially beneficial effect of calpain inhibition in diabetes. Therefore, we propose to study the role of calpains in the inflammatory response of the microcirculation during acute and chronic hyperglycemia. We will test the hypothesis that calpains cause vascular inflammation during hyperglycemia by studying whether: (1) calpain activity is abnormally increased; (2) inhibition of calpain activity prevents inflammatory events in the microcirculation; (3) increased calpain activity downregulates the eNOS enzyme leading to loss of physiologic levels of NO; (4) activation of calpains increases endothelial cell surface expression of pro-inflammatory adhesion molecules via upregulation of NF-kB activity. We will utilize the following in vivo and in vitro cell physiology techniques: intravital microscopy, culture of microvascular endothelial cells under static and flow conditions, NO measurements in vivo and in vitro, immunohistochemistry, Western blot analysis, and gel shift assays. By these means, the studies in this proposal will elucidate important and novel mechanisms underlying microvascular dysfunction in diabetes. This information should provide a framework for developing new therapeutic strategies for the treatment of the life-threatening disorder, diabetes mellitus.